7-chloro-6-demethyl-tetracycline fermentation

ABSTRACT

This disclosure describes a process for the production of 7chloro-6-demethyltetracycline by fermentative biosynthesis using strains of Streptomyces aureofaciens which impart a color to their whole harvest mashes such that a positive value of delta R is obtained when the values of the percent reflectance of a 1:200 aqueous dilution of the whole harvest mash at 460 m Mu , 540 m Mu , 560 m Mu and 660 m Mu are inserted in the following formula: delta R R540+ R560-1.1 R460-0.9 R660-2.

United States Patent Inventor John Andrew Growich, Jr. New City, N.Y.

Appl. No. 594,958

Filed Oct. 12, 1966 Patented Oct. 26, 1971 Assignee American Cyanamid Company Stamford, Conn.

Continuation of application Ser. No. 508,331, Nov. 17, 1965, now abandoned.

7-CHLORO-6-DEMETHYL-TETRACYCLINE FERMENTATION 2 Claims, 14 Drawing Figs.

U.S. Cl 195/80 Int. Cl C12d 9/00 Field of Search 195/80 [56] References Cited UNITED STATES PATENTS 2,878,289 3/1959 McCormick et al. 195/80 X 3,092,556 6/1963 Growich etalv 195/80 OTHER REFERENCES Chemical Abstracts, Vol. 62, 1965, pg. l5l07d, copy in Chemical Library Primary Examiner-Joseph M. Golian Attorney-Edward A4 Conroy, Jr.

PATENTEDum 2s l9?l 3,616,239

sum 01m 14 REFLECTANCE CURVE OF HOLE HARVEST MASH OBTAINED WITH STRAlN S604 PERCENT REFLECTANCE WAVELENGTH (MILLIMICRONS) A R= -33 INVENTOR.

JOHN ANDREW GROWICH,JR.

ATTORNEY PATENTEDUBI 2 s iSYI SHEET UZUF 14 STRAIN SIG?! REFLECTANCE CURVE OF WHOLE HARVEST MASH o'mmw WETH i 500 WAVELENGTH (MILLINIIGRONS) INVHNTOR.

ow 7 H G w D I N A07 N H I on B ATTORNEY PATENTEUHU 26 I971 3,616.2 39

SHEET 03 0F 14 OTAIND WBTH i STRAIN V62 PERCENT REFLECTANCE WAVELENGTH (MILLIMICRONS) AR L9 INVENTOR.

JOHN ANDREW GRfiJICHJR.

ATTORNEY PATENTEB 28 I971 3,616,239

SHEET 0% HF 14 OTAINE WWH STRAIN B740 PERCENT REFLECTANCE WAVELENGTH (MILLIMICRONS) INVIL'N'IUR. JOHN ANDREW GROWICH,JR.

ATTORNEY PAINTEDum 26 1911 SHEET USUF 14 REFLECTANCE CURVE OF WHOLE HARVEST MASH QBTAINE WETH S. STRAIN V|38 O ww 3 moz hom4mmm hzwomma WAVELENGTH (MILLIMICRONS) INVENTOR.

V: uwa w H R w 0 T W? T R A G I w j E R o W N A N H O v J B PATENTEDnm 26 ml 3,616,239

SHEET 05 0F 14 REFLEGTANGE GURVE 0F WHQLE HARVEST MASH @ETAQNEQ WW 5; zgg gammms STRAIN E475 PERCENT REFLECTANCE WAVELENGTH (MELLHMICRONS) A R INVENTOR.

JOHN ANDREW GROWICH,JR.

ATTORNEY PATENTEBum 2 s IBYI SHEET [170F 14 NGE CUVE OF WHOLE HARVEST MASH REFLECT OBTAI NE ITH S. STRAIN El3l| 0 w G v 4 3 WAVELENGTH (MILLIMICRONS) INVENTOR.

JOHN ANDREW GROW|CH,JR.

ATTORNEY 'PATENTEDncI 26 I9?! 3,616,239

SHEET 08 [1F 14 FEQPQ REFLECTANGE CURVE OF WHOLE HARVEST MASH OETAINEU WITH STRAIN EDI I23 PERCENT REFLECTANCE WAVELENGTH (MILLIMIGRONS) AR -6.l JNVENTOR.

JOHN ANDREW GROWICH,JR.

ATTORNEY PATENTEDUCT 26 I97! SHEET USUF 14 REFLECTANG GURVE OF WHOLE HARVEST MASH OTAINED WETH STRAIN E0204? O ww MOZFPOM EME .2m0mum WAVELENGTH (MILLIMICRONS) INVENTOR.

. Y mmvw, E H M w 0 w n 0 MW A w% n/ A M 1 W 0V1, JB

PATENTEUum 26 I97! SHEET lOUF 14 REFLECTANOE CURVE OF WHOLE HARVEST MASH OBTAINED WITH 3. AUREOFAOIENS STRAIN ED23|4 WAVELENGTH (MILLIMICRONS) INVENTOR. JOHN NDREW cowlcmm.

ATTORNEY PATENTEIJnm 26 I97! SHEET llUF 14 FIG?" REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH 8. AUREOF ACIENS STRAIN IE|352 O ww WAVELENGTH (MILLIMIGRONSY' AR=O.9

INVENTOR. JOHN ANDREW GROWICH,JR.

ATTORNEY PAIENTEDHBT 2 3,616,239

SHEET 12 0F 14 REFLECTANGE CUVE OF WHOLE HARVEST MASH OBTAINED WETH STRAIN IEI354 PERCENT REFLECTANGE WAVELENGTH (MILLIMICRONS) A R L8 INVENTOR.

JOHN ANDREW GROWICFLJR.

ATTORNEY PAIENTEHnm 251911 SHEET 13UF 14 REFLECTANGE CUVE OF WHOLE HARVEST MASH OBTAlNED WITH S. STRAIN IE|355 WAVELENGTH (MILLIMICRONS) AR=LI INVENTOR.

JOHN ANDREW GROWICH,JR.

ATTORNEY PATENTEDnm 26 m1 PERCENT REFLECTANCE SHEET 1: BF 1d Fflflfl REFLEGTANGE @URVE F WHQLE HARVEST MASH OTAHNEQ WHTH g STRAINIEZBOZ:

WAVELENGTH (MELLIMHCRONS) AR=4.7 INVENTOR.

JOHN ANDREW GROWICH,JR.

7-Cl-lLoRo-o-DEMETflYL-TETRACYCLINE FERMENTATION This application is a continuation-impart of my copending application Ser. No. 508,331, filed Nov. 17, 1965, now abandoned.

This invention relates to an improved process for the production of 7-chloro-fi-demethyltetracycline and, more particularly, is concerned with the selective production of 7- chloro-6-demethyletetracycline by fermentative biosynthesis using strains of Streptomyces aureofaciens which impart a color to their whole harvest mashes such that a positive value of SR is obtained when the values of the percent reflectance of a 1:200 aqueous dilution of the whole harvest mash at 460 m 540 m 560 my, and 660mg are inserted in the following formula:

The novel mutant strains of the present invention are strains of the species Streptomyces aureofaciens. The representative S. aureofaciens strains described below are direct descendants of the 7-chlorotetracycline-producing S. aureofaciens A377 soil isolate described in U.S. Pat. No. 2,482,055 to Duggar and deposited at the Northern Regional Research Laboratories, Peoria, 111. and indexed as NRRL 2209, derived by mutagenic processes known to the art, e.g., ultraviolet irradiation, nicotine, and nitrogen mustard. Typical mutant strains of S. aureofaciens which possess the unique properties described herein have been designated by me as 113-1352, lE-1354, 115-1355 and 115-2803. Viable representative cultures of the novel mutant strains of the present invention have been deposited at the Northern Regional Research Laboratories, Peoria, 111. and have been assigned the following accession numbers:

Strain NRRL No. -1352 3201 18-1354 3202 15-1355 3203 18-2803 3235 It will be understood, of course, that mutants which possess this unique property of imparting a specific color to their whole harvest mashes may be derived from these strains by conventional procedures. These mutants may vary somewhat in general morphological and physiological characteristics as do the various strains of the S. aureofaciens species. It is also to be expected that 7-chloro-6-demethyltetracycline-producing strains of S. aureofaciens of the invention, i.e., with the required positive 8R value, may be found in nature or derived from presently isolated strains of S. aureofaciens by mutagenic methods well known to the art. The strains of the present invention display the fundamental characteristics of the species S. aureofaciens and may be readily identified as members of this species although differing from previously described strains of S. aureofaciens, not only in pigmentation on solid media, but also in the colors of the whole harvest mashes obtained therewith.

The antibiotic 7-chloro--demethyltetracycline was first described and claimed, together with methods of its production, in the McCormick et al. U.S. Pat. No. 2,878,289 wherein a 7-chloro-6-demethyletracycline-producing strain of S. aureofaciens was cultivated in a fermentation medium. That patent also discloses that other tetracyclines, such as 6- demethyltetracycline, 7-chlorotetracycline and tetracycline are produced at the same time in a fermentation medium when the fermentation conditions, including the micro-organism therein disclosed, are used. Additionally, that patent discloses that 7-bromo-6-demethyltetracycline is a concomitant product when bromine is a constituent of the fermentation medium. Further evidence of the production of more than one tetracycline in a fermentation medium in which demethylchlortetracycline is produced may be found in the Perlman et al. U.S. Pat. No. 3,028,311 and the Neidleman U.S. Pat. No. 3,061,552 wherein so-called G-methylation inhibitors are present in a fermentation medium in which a 7- chlortetracycline-producing strain of S. aureofaciens is used.

Additional disclosures of the production of 7-chloro-6- demethyltetracycline by cultivating 7-chlortetracycline producing strains of S. aureofaciens in the presence of 6- methylation inhibitors are shown in the Goodman et al. U.S. Pat. Nos. 3,019,172 and 3,127,328. These processes are also faced with the problem of concomitant formation of other tetracyclines. Thus, in each a mixture of 7-chlortetracycline, tetracycline, 7-chloro-6-demethyltetracycline and 6- demethyltetracycline is produced.

The concomitant production of o-demethyltetracycline (or other tetracyclines) is objectionable when 7-chloro-6- demethyltetracycline is the principal product being sought. While the specifications of pharmaceutical quality 7-chloro-6- demethyltetracycline permit small quantities of -demethyltetracycline to be present, the presence of any sizable quantity of fi-demethyltetracycline is objectionable. The presence of these two antibiotics in the same fermentation mash in any sizable amounts also involves diflicult problems of separation in the refining or extraction procedures when 7-chloro-6- demethyltetracycline is the desired product, It is possible, of course, to extract the two antibiotics from the fermentation mash and by selective refining procedures to effect a separation of the antibiotics. However, the refining procedures for effecting separation of the antibiotics are difficult and usually involve some loss in total antibiotic potency. Moreover, in those instances where 7-chloro-6-demethyltetracycline is the principal product of the fermentation, o-demethyltetracycline is unwanted and is customarily removed and discarded. Hence, any method that results in the increased production of 7-chloro-6-demethyltetracycline and the le$ened concomitant production of o-demethyltetracycline constitutes a marked contribution to the art of antibiotic fermentation.

It is indeed surprising that the novel mutant strains of Streptomyces aureofaciens of the present invention will produce, in a conventional fermentation, 7-chloro-6- demethyltetracycline with no more than 5 percent of 6- demethyltetracycline as a concomitant product. Furthermore, there is no detectable production of tetracycline or 7 chiorotetracycline. The discovery of these strains obviates the need for costly and difficult extraction and purification procedures, since the crystals of 7-chloro-6-demethyltetracycline obtained are of pharmaceutical quality. Obviously the discovery of these strains denotes a marked contribution to the art of producing the highly effective antibiotic 7-ch1oro-6- demethyltetracycline.

In general, these new strains and the strains which are presently known in the art which produce 7-chloro-6- demethyltetracycline in conventional media may be divided into three categories: l) S. aureofaciens strains ATCC 12551, 12552, 12553 and 12554 which are described in U.S. Pat. No. 2,878,289 McCormick et al. and which produce varying amounts of 7-chloro-o-demethyltetracycline, o-demethyltetracycline, tetracycline and 7-chlorotetracycline, (2) S. aureofaciens strains V-138, 13-475, E-1311, ED1723, ED-2047 and E.D2314 which are disclosed in the following patents: V-138 in US. Pat. No. 3,037,916 (Goodman), E475, 13-1311, ED-1723 and ED-2047 in U.S. Pat. No. 3,050,446 (Goodman), ED-2314 in U.S. Pat. No. 3,145,154 (Goodman); and which produce varying amounts of 7-chloro- -demethyltetracycline and o-demethyltetracycline to the virtua! exclusion of methylated tetracyclines; and (3) the new strains of the present invention, e.g., 1E-l352, -1354, 113-1355 and 115-2803, which produce 7-chloro-6-demethyltetracycline to the virtual exclusion of all other tetracyclines.

The novel mutant strains of the present invention may be distinguished from the two groups of prior art strains by a mathematical interpretation of the spectrophotometric reflectance curves of their respective whole harvest mashes. More precisely, when the percent reflectance of diluted samples (1:200 aqueous) of the whole harvest mashes of good yield (such as the examples set forth hereinafter) of the novel strains of the present invention is measured at specific wavelengths (460 m 540 m 560 my. and 660 my.) and the percentage reflectances in these specific wavelengths are inserted in the formula:

8R=R +R l l R 0.9R 2.0

a definite pattern appears. Each the representative strains of the present invention, 15-1352, lE-l354, lE-l355 and lE2803, produce a positive 6R value (greater than while each of the representative strains of the prior art produce a negative 8R value (less than 0). The letter R denotes the percent reflectance value at some particular wavelength, for example, R is the percent reflectance value at a wavelength of 560 my FIGS. 1 through 14 of the drawings are spectrophotometric reflectance curves obtained when l-centimeter glass cells are filled with a diluted (1:200) whole harvest mash sample which is taken from the harvest fermentation broth of the novel mutant strains of the present invention as well as the additional previously described prior art strains listed in table I. The spectrophotometric reflectance curve of a material constitutes a permanent record that does not require maintenance of a sample. Furthermore, the units in which the curve is expressed are internationally understood and accepted as a means of measuring color. In FIGS. 1 through 14, the wavelength of light in rnillimicrons is plotted as the abscissa against the reflectance as the ordinate. The wavelength of light has been adopted internationally as the fundamental standard of length to which all other standards of length are referred. These spectrophotometric reflectance curves were determined with a recording spectrophotometer known as the General Electric Hardy Recording Spectrophotometer with the sample backed with magnesium oxide, the reference standard being magnesium oxide, and with the specular component excluded. The curves are recorded on General Electric Co. Chart paper NP 62581.

With regard to the aforementioned spectrophotometric determination it should be noted that dilution of the whole harvest mash is necessary to produce the mathematical results as stated. If the whole harvest mash is used undiluted, the difference in the 8R value between the new and prior art strains will not be evident. it is only when the whole harvest mash is diluted with water at the rate of one part of whole harvest mash to 199 parts of water that the unusual results of a positive 5R value for the new strains and a negative 5R value for the prior art strains becomes evident.

A whole harvest mash is the untreated mash obtained after a fermentation employing the following medium:

Starch-45 grams CaCO 10.5 grams NH Cll .5 grams Cottonseed flour-45 grams Yeast l .5 grams Water-q.s. to-l000 ml.

Lard oil3% (Vol/vol. of medium) has proceeded to the point where biosynthesis of the primary antibiotic product has stopped for all practical purposes. Generally, under the conditions as set forth hereinbelow, the antibiotic potency of the fermentation mash, as may be conventionally determined, ceases to rise appreciably after the fermentation has proceeded for from about 140 to about 160 hours.

The novel mutant strains of S. aureofaciens of the present invention which produce virtually no other tetracycline except 7-chloro-6-demethyletracycline possess essentially the same morphological characteristics as do the strains which concomitantly produce 7-chlorotetracycline, tetracycline, 7- chloro--demethyltetracycline and G-demethyltetracycline and those strains which produce 7-chloro-6-demethyltetracycline and a substantial proportion of odemethyltetracycline, but differ among themselves in the same general manner that these prior art strains differ from each other as has been described in a number of scientific papers which have been published. The data appearing below will serve to further Color observations S. aureofacgmg 8P4 Agar: Six-day incubation at 6.

Single colonies Major colony Minor colony Strain type I type 11 Mass growth 113-1352. Chocolate brown to Ebony brown Chocolate brown deep brown to dark brown mahogany. mahogany. 115-1354. Oak brown to deep Dark brown Copper brown to brown. mahogany. chocolate. 113-1355. Deep brown to .do Dark brown chocolate brown. mnh ogany. 1E-2803- ..do do Cocoa brown.

1 Colors here and elsewhere throughout this specification are according iipltlhepolor Harmony Manual, Third Edition, Container Corporation of once.

FORMULATlON OF AP4 AGAR Sucrose MgSO; 7H

KH PO. Htlz o-i Cornsleep. Bacto agar Water. q.s.... Post Sterilization pH is 6.3

grams The novel 5. aureofaciens strains of the present invention were differentiated from S. aureafaciens A-377 (NRRL 2209) by observation of growth characteristics on various media incubated at 26.5 C. until maturity.

(1) GLYCEROL ASPARAGINE BEEF EXTRACT AGAIL Glycerol, grams l. L-asparagine, grams. Beef extract, grams KHzPOi, grams Becto agar, grams Distilled water, (1.5. 100.0 ml. Adjust to pH 7.0 with 50% RON. Post sterilization p11 is 7.1.

Streptomyccs aureofaciem Strain 113-1354 Strain 113-1352 Growth. Scanty to thin, light mus- Moderate to abundant,

turd tan to mustard light amber. tan. Aerial hyphae Thin becoming fair to moderate, 5cb.

Fair to moderate pussywillow grey to ushcs.

Sporulationm" Fair to moderate.. Fair to moderate. Difiusiblc ellow Yellow.

pigment. Reverse Light grey becoming olive Light amber.

green.

Strain 1E-1355 Strain 5-604 Growth Moderate, light mustard Abundant, venetian red.

tan to mustard tan.

Aerial hyphae Thin, white becoming Abundant, white to rose Color Harmony Manual, Third 1 Color chip code according to the no assigned color name.

Edition, Container Corp. of America;

(2) DEXTRIN OZAPEK-DOX AGAR Dextrin, grams 1. NaNOa, grams. 0.2 K2HPO4, grams 0. 1 MgSOr-7Hz0, grams. 0.05 KCl, grams 0. FGSO4-7H2O, grams.. 0.001 Bacto agar, grams 1. 5

Distilled water, q.s. 100 ml.

Post sterilization pH is 7.2.

Streptomyces aureofacims Strain 1E-1352 Strain 1E-1354 Growth Confluent, thin, translu- Confluent, thin, translucent, colorless. cent, colorless.

Aerial hyphae.. None.. None. Spor'ulation. ...do.. Do. Diffusible pig- .....do Do.

ment. Reverse Translucent Translucent.

Strain 1E-1355 Strain S4104 Growth Confluent, thin, translu- Sparse, hyaline.

cent, colorless. Aerial hyphae None None. Sporulation ..do Do. Difiusible pig- ...do. Do.

ment. Reverse Translucent Apigmentous.

Strain A-377 Strain 1E-2803 Growth Profuse Confluent, thin, transparent, colorless. Aerial hyphae.. Abundant, lead grey, None.

water white surface globules. Sporulation- Abundant Do. Difiusitble pig- Slight, pale yellow Do.

men Reverse Apigmentous Translucent.

3) AP4 Adan as DESCRIBED anoint) '4 Streptomyces aureofaciens Strain 113-1354 Strain lE-l352 Profuse, copper brown to to dark rose brown. Thin, pussywillow grey.

grey. Sporulation Fair, complete Thin, complete. Difiusible Reddish-brown Amber to light red pigment. brown. Reverse Chocolate brown to dark Copper brown to dark rose brown. rose brown.

Strain 113-1355 Strain S4504 Growth Profuse, dark brown Profuse.

mahogany. Aerial hyphae Moderate, ashes Altiaundant, dark rose upe. Sporulation Fair Very abundant, uniform. Diffusible Red-brown Very concentrated. Deep pigment. brown to deep brown mahogany. Reverse Dark brown mahogany... Dark brown mahogany.

Strain A-377 Strain 115-2803 Growth Profuse Abundant, deep brown mahogany. Aerial hyphae. Abundant, beaver None. Sporulation.... Very abundant, uniform Do. Difiusible Light greenish-yellow..... Reddish brown.

pigment. Reverse Covert brown Deep brown mahogany.

(4) Q4 CORNSTEEP AGAR Cornsteep, grams 9 Sucrose, grams l 0.25

Crude agar, grams 30 Water (1.5. 1,000 milliliter s:

Aerial hyphae. Fair to moderate, white becoming pussywillow y.

Profuse, chocolate.

Sporulation..... Thin to fair Profuse.

Difiuslble Amber Burgundypigment.

Reverse Burgundy Do.

5 Strain A-377 Strain 1E-2803 Growth Excellent, pale yellow Abundant, copper brown becoming dark rose own.

Aerial hyphae.. Profuse, dark brown Fair to moderate, ashes.

Sporulation. Profuse Thin to fair.

Difiusible Orange-brown Dark red brown.

10 pigment.

Reverse Orange to orange-yellow... Burgundy.

(5) OTHER AGAR MEDIA Strcptomyccs aureofaciens Medium Strain lE-l352 Strain lE-1354 Nutrient Poor to fair growth; dark Thin growth; deep brown agar. spice brown to chocolate to dark brown mahogany.

brown. N o aerial hyphne. No aerial hypbac. Faint Light amber soluble pigred-brown soluble pigment. Reverse: Chocolate ment. Reverse: Deep brown. brown to dark brown mahogany.

Glucose Abundant growth; copper Abundant growth; maple to asparabrown. Abundant aerial light brown. Abundant gine hyphae; pussywillow aerial hypbae ashes. meat grey becoming ashes. Moderate to abundant extract Sporulation: abundant. sporulation. Deep yellow agar. Deep amber soluble pigsoluble pigment. Reverse:

ment. Reverse: Copper M nple to light brown.

brown.

Purple Pronounced growth collar; Pronounced growth collar;

milk. taupe brown to rose taupe brown to rose brown. Little significant brown. Little significant pH change. False alkaline pH change. False alkaline color reaction due to pigcolor reaction due to pigment diffusion. ment diffusion.

pH 6.55 at 2 weeks. pH 6.8 at 2 weeks.

Potato Profuse, moist crenulated Profuse, moist crcnulated slants. growth; chestnut brown growth; covert brown to to chocolate brown bcluggage tau becoming coming ebony. Rose chocolate brown. Rose taupe soluble pigment. taupe soluble pigment. No aerial hypbae. No aerial hyplnre. Sfreptomyccs aurcofaciens Medium Strain 112-1355 Strain S-GOt Nutrient Thin growth; Dark spice Poor growth. Taupe brown agar. brown to chocolate to dark brown. N o aerial brown. No aerial liypinie. hyphne. Reverse: Taupe Very light amber soluble brown. Rcddish brown pigment. Reverse: soluble pigment. chocolate brown.

Glucose Abundant growth; cocoa Abundant growth. Heavy asparbrown to copper brown. nerlnl hyphae mottled. agine- Abundant aerial hyphae; Rose taupe to fawn to meat ashes. Abundant sporucnmcl. Sporulntion:

extract lation. Deep yellow abundant. Reverse:

agar. soluble pigment. Reverse: tnupc brown. Rcddish cocoa brown to copper brown soluble pigment. brown.

Purple Pronounced growth collar; Slight deep red mahogany milk. taupe brown to rose growth collar. Little slgbrown. Little significant nilicnnt pH change nor ll change. False alkaapparent pcptonization.

mo color reaction due to Slight false alkaline color pigment diffusion. pll at reaction due to diffusion 2 weeks. of soluble pigment.

Potato Profuse, moist crenulated Profuse, moist smooth slants. growth; copper brown to noduluted growth; (lurk deep brown becoming brown mahogany with chocolate brown. Rose peach tan trace. Aerial 5 5 taupe soluble pigment. hyphae; none to abundant N0 nerialhyphae. becoming white to camel.

SporulatiOn abundant in areas of heavy nerinl hyphae. Chocolate to chocolate brown soluble pigment. b0 Streptomyces aureofaciens Medium Strain A-377 Strain 1E"2803 Nutrient Fair growth. N o aerial Poor to fair confluent agar. hyphac. Reverse: Polc growth; clove brown to yellow. Yellow to light sepia brown. N o aerial brownish yellow soluble hyphac. Tawny diffusiblc pigment. pigment tinted reddish brown. Reverse: Clove brown to sepia brown.

Glucose Fair growth. Aerial hyphae Abundant growth; deep Asparwhite becoming increasbrown. Abundant, comagmemgly grey with increase in pact aerial hyphae, ashes. meat spore formation. Reverse: Abundant sporulation.

extract Light yellow. Light yellow Light to moderate reddish agar. soluble pigment. brown diffusible pigment becoming amber. Reverse: Deep brown becoming dark brown mahogany.

Purple Slight white to pale yellow Pronounced growth collar;

milk. growth collar. Little sigtaupe brown to dark rose nifieant pH change nor brown. Little significant apparent peptonization in 15 days.

pH change. False alkaline color reaction due to pigment diffusion. pH 6.75 at 2 weeks.

Potato Profuse moist smooth nod- Profuse, moist, orenulatcd (6) lii'iC-ROSCOPIC OBSERVATIONS Glucose asparegine beet extract agar S. uureofacicm Q4 agar Strain 1E-l352:

Mycelium- Flexuous, continuous, Flexuous, continuous,

branched. Diameter branched. Diameter 0.8 to 1.2 4. 0.8 to 1.215. Spores Spherical to ovoidal. Spherical to ovoidal Diameter 0.8 to 1.6;. Diameter 0.8 to 1.5 Strain iE-IBM:

Mycelium. Flexuous, continuous, Flexuous, continuous, branched. Diameter branched. Diameter 0.8 to 1.2 4. 0.8 to 1.2a. Spores Spherical to ovoidal. Spherical to ovoidal.

Diameter 0.8 to 1.5 1. Diameter 0.8 to 1.5;. Strain iii-1355:

Myceliurm. Flexuous, continuous, Flexuous. continuous, branched. Diameter branched. Diameter 0.8 to .2 4. 0.8 to 1.2;. Spores Spherical to ovoldal. Spherical to ovoidal.

Diameter 0.8 to 1.6;:- Diameter 0.8 to 1.5;. Strain 5-604: 1

Myceli Flexuous, continuous, Flexuous, continuous, branched. Diameter branched. Diameter 0.8 to 1.0 4. 0.8 to 1.2 4. Spores Spberoidal to ovoidai. Spheroidal to ovoidal.

Diameter 1.5 to 2.0a. Diameter 1.6 to 2.0 Strain A-377:

Mycelium. Flexuous, continuous, branched. Diameter Flexuous, continuous, branched. Diameter Spores spheroidal to ovoidal. spheroidal to ovoidal. Diameter 1.6 to 2.0 Diameter 1.6 to 2.0.1:.

Strain 115-2803:

Mycelinm. Flexuens, continuous, Fleruous, continuous, branched. Diameter branched. Diameter 0.8 to 1.2a. 0.8 to 1.2

Spores Spherical to ovoidal. Spherical to ovoidal.

Diameter 0.8 to Leo. Diameter 0.8 to 1.541.

Norn.-Mycelial and spore morphology as viewed throu h the light microscope, is apparently similar for strains 115-1352, 113-! 4, iii-i855 and 1E-2803 as for strains A-377 and 8-004. However, the diameters for The conditions of the fermentation are generally the same as for the presently known methods of producing chlortetracycline by fermentation. That is, the fermentation medium contains the usual assimilablc sourccs of carbon, nitrogen and inorganic salts. Suitable nutrient substances include starch, dextrose, cane sugar, glucose, molasses, soy bean meal, milk solids, yeast, meat extracts, pcptonc, urea, comstccp liquor, cottonseed meal, distillers' solublcs, glyccridc oils, fish meal and other conventional substances. The inorganic salts include such things as calcium carbonate, ammonium sulfate, ammoniun. chloride, and salts of the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

The other general conditions of the fermentation such as hydrogen ion concentration, temperature, time, rate of aeration, preparation of the inoculum, sterilization, inoculation and the like are conventional and may be similar to those for the production of 7-chloro-o-demcthyltetracyclinc shown in US. Pat. No. 2,878,289 (McCormick et al.).

The recovery of the 7-chloro-6-dcmethyltetracyclinc from the fermentation liquor is conventional and need not be described, as numerous methods for the recovery of this product from fermentation liquors have already been published.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 A fermentation medium was prepared which contained the following ingredients:

Starch-45 grams CaC 10.5 grams NH,C1 L grams Cottonseed flour-45 grams Yeast-1.5 grams Water-q.s. tol 000 milliliters This medium was dispensed in appropriate amounts into flasks containing 3 percent (vol./vol. of medium) of lard oil and then sterilized. These flasks were then divided into 14 approximately equal groups. Each group was then inoculated with a vegetative inoculum of one of the following strains of S. au-

reofaciens.

5-604 (ATCC 12551) S-l07l (ATCC12S52) V-62 (ATCC12S53) 3-740 (ATCC [2554) V-l38 ED-l723 iii-i352 (NRRL3201) .54 Hi er-e202)... ii-i355 NRRL3203) Iii-2803 (NRRL3235) All of the flasks were then incubated on a rotary shaker with a Zia-inch circulator motion at 185-200 r.p.m. at 25 C. for I hours. Samples were withdrawn, diluted with 199 parts of water, and the reflectance curves determined as set forth hercinabove. The results are set forth in FIGS. 1 through 14. The harvest mash assays are recorded in table I below. These assays were performed using the following methods: Assay No. i: This assay is based upon the difference in the rates of conversion of 7-chloro-6-demethyltetracycline and 6- dcmcthyltctracycline to their respective anhydro forms by an acid catalyzed, first order reaction. The degree of conversion to the anhydro form depends upon time, temperature and acid strength involving a reaction which results in a decrease in absorbance at 368 my. and an increase in absorbance at 440 m Assay No. 2: This assay is based upon the same basic principle as assay no. 1 except that in this case the sample is treated with acid to degrade all tclracyclincs present to the anhydro form in one instance and compared to a sample treated with water in the second instance. Assay No. 3: The assay figures in this column were determined by a conventional paper chromatographic procedure using the system 0.3 M sodium phosphate nbutyl acetate, giving an R of 0.30 for o-demethyltetracycline. Three similar systems, published in the Journal of the American Chemical Society, Vol. 79, pp. 4561-4563 (1957), (McCormick ct al.) are listed below.

System o-bemcthylictracyclinc 0.3 M Sodium Phosphateln-buianol 0.30

in" Mac llvainc': Buffer/ethyl acetate 0.27

(pH 4.7) 0.: N n,P0,, 0.1% ccncoou an 0.22

TABLE I Assay N0. 1 No. 2 N0. 3

Total DMTC, 'y/mL, tetraquantitative S. aureofaciens DMCTC, cyclines, paper Percent strain No. 7/1111. 'ylml chromatogram DMTC What is claimed is: 1. The process of producing 7-chloro-6-demethyltetracycline which comprises cultivating a strain of Slreptomyces aureofaciens which produces 7-chloro'6-demethyltetracycline to the substantial exclusion of other tctracyclines selected from the group consisting muthose having accession numbers NRRL 3201, NRRL 3202, NRRL 3203 and NRRL 3235, and mutants thereof under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts until substantial quantities of 7-chloro-o-demethyltetracycline are produced, said 7- chloro-6-demethyltetracycline-producing strain of Streptomyces aureofaciens being characterized by its ability to impart to a ZOO-fold aqueous dilution of the whole harvest mash a color characterized by a reflectance curve, when plotted linearly, wherein the values of the percent reflectance at 460 mp, 540 mp., 560 m and 660 mp. when substituted into the formula:

6R=R +R l l R ,,0.9R 2.0

give a positive value for SR.

2. A process according to claim 1 in which the amount of 6- demethyltetracycline produced is no more than 5 percent by weight of the amount of 7-chloro-o-demethyltetracycline produced. 

2. A process according to claim 1 in which the amount of 6-demethyltetracycline produced is no more than 5 percent by weight of the amount of 7-chloro-6-demethyltetracycline produced. 